The heart (FIG. 1A) is the muscular organ that pumps blood through the blood vessels of the circulatory system. In a healthy mammalian heart, composed of right and left atrium and right and left ventricles, blood flows only one way through the heart because the heart valves prevent backflow. The heart is enclosed in a protective sac, the pericardium, which also contains a small amount of fluid. The wall of the heart is made up of three layers: epicardium, myocardium, and endocardium.
The sinoatrial node (often abbreviated SA node or SAN; also commonly called the sinus node and less commonly the sinuatrial node) is the pacemaker of the heart and is responsible for the initiation of the heartbeat. It is located at the junction of the vena cava superior and the atrium, and measures about 5 mm by 2 cm. It spontaneously generates an electrical impulse, which after conducting throughout the heart, causes the heart to contract. Although the electrical impulses are generated spontaneously, the rate of the impulses (and therefore the heart rate) is modified by the nerves innervating the sinoatrial node, located in the right atrium (upper chamber) of the heart.
The atrioventricular (AV) node is a part of the electrical conduction system of the heart that coordinates the contractions of the heart chambers. It electrically connects atrial and ventricular chambers (FIG. 1B). The AV node is an area of specialized tissue between the atria and the ventricles of the heart, specifically in the posteoinferior region of the interatrial septum near the opening of the coronary sinus, which conducts the normal electrical impulse from the atria to the ventricles. The AV node is quite compact (˜1×3×5 mm). It is located at the center of Koch's triangle—a triangle enclosed by the septal leaflet of the tricuspid valve, the coronary sinus, and the membraneous part of the interatrial septum.
The distal portion of the AV node is known as the Bundle of His. The Bundle of His splits into two branches in the interventricular septum, the left bundle branch and the right bundle branch. The left bundle branch activates the left ventricle, while the right bundle branch activates the right ventricle. The two bundle branches taper out to produce numerous Purkinje fibers, which stimulate individual groups of myocardial cells to contract.
Funny current (or funny channel, or If or IKf, or pacemaker current) refers to a specific current in the heart. First described in the late 1970s in Purkinje fibers and sinoatrial myocytes, the cardiac pacemaker “funny” current has been extensively characterized and its role in cardiac pacemaking has been investigated.
The funny current is highly expressed in spontaneously active cardiac regions, such as the sinoatrial node, the atrioventricular node and the Purkinje fibers of conduction tissue. The funny current is a mixed sodium-potassium current that activates upon hyperpolarization at voltages in the diastolic range (normally from −60/−70 mV to −40 mV). When at the end of a sinoatrial action potential, the membrane repolarizes below the If threshold (about −40/−50 mV), the funny current is activated and supplies inward current, which is responsible for starting the diastolic depolarization phase (DD). With this mechanism, the funny current controls the rate of spontaneous activity of sinoatrial myocytes and thus the cardiac rate.
The molecular determinants of the pacemaker current belong to the Hyperpolarization-activated Cyclic Nucleotide-gated channels family (HCN) of which 4 isoforms (HCN1-4) are known to date. Based on their sequence, HCN channels are classified as members of the superfamily of voltage-gated K+ (Kv) and cyclic nucleotide-gated (CNG) ion channels.
In simple terms, the electrical signals that control the heartbeat can be described as follows. The electrical impulse starts in the SA node. The electrical activity spreads through the walls of the atria and causes them to contract. This forces blood into the ventricles. The AV node acts like a gate that slows the electrical signal before it enters the ventricles. This delay gives the atria time to contract before the ventricles do. After this delay, the stimulus diverges and is conducted through the left and right bundle of His to the respective Purkinje fibers for each side of the heart, as well as to the endocardium at the apex of the heart, then finally to the ventricular epicardium. The His-Purkinje Network of fibers sends the impulse to the muscular walls of the ventricles and causes them to contract. This forces blood out of the heart to the lungs and body. The SA node fires another impulse and the cycle begins again.
Many things can go wrong with the heart resulting in irregular beating. Sick sinus syndrome—also known as sinus node disease or sinus node dysfunction—is the name for a group of heart rhythm problems (arrhythmias) in which the sinus node—the heart's natural pacemaker—doesn't work properly.
An artificial pacemaker is a medical device that uses electrical impulses, delivered by electrodes depolarizing the heart muscles, to regulate the beating of the heart. The primary purpose of the artificial pacemaker is to maintain an adequate heart rate, either because the heart's natural pacemaker is not fast enough, or because there is a block in the heart's electrical conduction system. Modern pacemakers are externally programmable and allow a cardiologist to select the optimum pacing modes for individual patients. Some combine a pacemaker and defibrillator in a single implantable device. Others have multiple electrodes stimulating differing positions within the heart to improve synchronization of the higher (atria) and the lower chambers (ventricles) of the heart.
Although artificial pacemakers have saved many lives, they are not a perfect solution. In particular, they are not hormone responsive, are subject to mechanical and/or electrical failure, need battery replacement and can be disrupted in strong magnetic fields or in therapeutic radiation settings. Further, infection is always a hazard, as is pacemaker-mediated tachycardia, suboptimal atrioventricular (AV) synchrony, and several other types of pacemaker induced dysrhythmias. Therefore, there are ongoing efforts to develop a more natural pacemaker.
Biological pacemakers, generally intended as cell substrates able to induce spontaneous activity in silent tissue, represent a potential tool to overcome the limitations of electronic pacemakers. Efforts to develop natural pacemakers for use in place of artificial pacemakers have generally taken one of two approaches.
One approach is to convert beating myocardium into pacemaker cells in situ via genetic manipulations (i.e., direct reprogramming). In this regard, the early key transcription factor TBX3 provided promising results, but led to cells with incomplete pacemaker characteristics.
Another approach is to use embryonic or induced pluripotent stem cells (so called IPS cells) that have been programmed to form pacemaker cells, and then replace or supplement AV cells with these newly programmed AV-like cells.
In a study, Jung et al. attempted to generate pacemaker cells by up-regulation of TBX3 in induced pluripotent stem cells (iPSCs). Hashem et al. (2013) indicated that SHOX2 regulates the pacemaker gene program in embryoid bodies. Bakker et al. (2012) attempted to reprogram terminally differentiated cardiomyocytes towards pacemaker cells by upregulation of TBX3. In another study Kapoor et al. (2013) attempted to generate pacemaker cells by overexpression of TBX18 in adult cardiomyocytes. Hu et al. (2014) tried to convert cardiomyocytes into pacemaker cells by upregulation of Tbx18.
Despite several attempts in the past to generate cardiac pacemaker cells, there are up to now no correctly functional biological pacemaker cells available for clinical application derived from undifferentiated adult autologous stem cells. It only has been shown before that cells like embryonic cells or IPS cells could be induced by manipulation of certain genes to obtain some features that are present in pacemaker or in other cells of the cardiac conduction system. The expression of a single transcription factor alone was not able to switch on the complete respective regulatory differentiation pathway towards pacemaker or Purkinje cells. Attempts to generate pacemaker cells in the past failed to mimic the appropriate physiological functionality and morphological properties of natural cardiac pacemaker cells.
The work described herein, takes this research to a new and higher state: The induction of differentiation of adult, unmodified, fresh uncultured cells (herein also called regenerative cells) from the patient's own tissue into non-contractile cardiomyocyte cells with morphological and functional structure and features of natural pacemakers and Purkinje cells has not been achieved before.